The objective of this proposal is to delineate the physiological roles of eukaryotic RNA unwinding (single-strand specific) proteins. Several such proteins from calf thymus and human cells have previously been shown to facilitate the in vitro renaturation of RNA molecules, suggesting an involvement of these proteins in aspects of RNA physiology where conformational interchange may occur. These aspects include transcription, nucleocytoplasmic transport of RNA molecules, translation, and reverse transcription. Using nucleic acid affinity chromatography, we propose to isolate RNA unwinding proteins from yeast, where the availability of mutant strains offers the possibility of unambiguously determining the physiological roles of these proteins, and from dinoflagellates, where the search for such proteins will be simplified by the small number of nucleic acid-interacting proteins in these organisms. Proteins isolated from these unicellular eukaryotes, as well as those already isolated from mammalian cells, will be tested for RNA-unwinding activities in template conformation-dependent cell-free assays which approximate the in vivo processes of reverse transcription, transcription and translation. The development of an in vitro assay for RNA transport, using artificial lipid bilayer membranes, is also envisioned. Finally, the mechanism by which unwinding proteins facilitate RNA conformation interchange will be further studied, using spectrofluorimetry, ultraviolet absorbance spectrophotometry, and other physical and chemical techniques.